Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis.Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors. (C) 2011 Elsevier B.V. All rights reserved.

Description of Leishmania (Leishmania) forattinii sp. n., a new parasite infecting opossums and rodents in Brazil

A new parasite species of Leishmania is described, L. (Leishmania) forattinii sp. n., which was isolated from a pooled triturate of liver and spleen of a opossum (Didelphis marsupialis aurita) and from skin samples from a rodent (Proechmys iheringi denigratus), captured in primary forest on the Atlantic Cost of Brazil. Our results on the basis of biological and molecular criteria indicate that this taxonomically distinct parasite ias a new species of the L. mexicana complex, but closely related to L. (L.) aristidesi Laison & shaw, 1979, as revelated by phenetic and phylogenetic numerical analyses of the enzyme data. L. forattinii was clearly distinguishable from other Leishmania species of the genus usisng enzyme electrophoresis, monoclonal antibodies, molecular karyotypes, analysis of restriction enzyme digestion patterns of kinetoplast DNA (kDNA), as well as the use of kDNA hybridization procedures.

Characterization of an unidentified Sarcocystis falcatula-like parasite from the South American opossum, Didelphis albiventris from Brazil

An unidentified isolate of a Sarcocystis falcatula-like parasite was obtained from the lungs of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris from Brazil. Four captive budgerigars fed sporocysts from the opossum intestine died of acute sarcocystosis 8, 10, and 12 days after oral inoculation (DAI); one budgerigar was killed 12 DAI when it was lethargic. Schizonts and merozoites found in the lungs of the budgerigars reacted mildly with polyclonal S. falcatula antibody. The parasite was isolated in equine kidney cell cultures inoculated with lung tissue from a budgerigar that was killed 12 DAI. Two budgerigars inoculated subcutaneously with 100,000 culture-derived S. falcatula merozoites developed acute sarcocystosis and S. falcatula-like schizonts were found in their lungs 15 and 16 DAI. Four budgerigars kept as unfed controls in the same environment remained free of Sarcocystis infection. The parasite underwent schizogony in African green monkey kidney cells and bovine turbinate cells. Merozoites divided by endopolygeny, often leaving a residual body. Polymerase chain reaction studies using primers JNB33/JNB54 and Hinf I and Dra I digestion indicated that the isolate was not S. falcatula. Results of this study indicated that the South American opossum, D. albiventris, is a definitive host for yet another S. falcatula-like parasite.

SEASONAL SPACE USAGE BY THE INTRODUCED PHARAOHS ANT, MONOMORIUM-PHARAONIS (L) (HYM, FORMICIDAE), IN INSTITUTIONAL SETTINGS IN BRAZIL AND ITS RELATION TO OTHER STRUCTURAL ANT SPECIES

In an institutional setting in Brazil, the pharaoh's ant, Monomorium pharaonis was found to be the dominant and most abundant ant present throughout the year. The only other regularly collected ant was the exotic Tapinoma melanocephalum. However, M. pharaonis dominated bait station visitations, although locally concentrated, with other species restricted to regions in which M. pharaonis was not found.

Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses. (c) 2005 Elsevier B.V. All tights reserved.

Purine nucleoside phosphorylase: A potential target for the development of drugs to treat T-cell- and apicomplexan parasite-mediated diseases

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell and Apicomplexan parasites-mediated diseases.

Density and population size of mammals introduced on a land-bridge island in southeastern Brazil

The introduction of alien species is one of the main threats to the conservation of native species, especially in island ecosystems. Here, we report on the population growth of 15 species of mammals introduced in 1983 on the island of Anchieta, an 828 ha land-bridge island in southeastern Brazil. We estimated the density of mammals through 296 km of line transect census. Five species introduced became extinct (coypu, brocket deer, six-banded armadillo, nine-banded armadillo, maned three-toed sloth); six became over-abundant (marmoset, coati, agouti, seven-banded armadillo, and capybara); one has a stable population (capuchin monkey). Anchieta Island has the highest density of mammals in the entire Atlantic forest (486.77 ind/km(2)), especially nest predators (232.83 ind/km(2)) and herbivores (253.58 ind/km(2)). Agoutis (Dasyprocta spp.) and marmosets (Callithrix penicillata) were, by far, the species with the highest population growth. The high density of mammals in this island may have strong consequences for plant recruitment and bird diversity.

Mebendazole treatment against Anacanthoras penilabiatus (Monogenea, Dactylogyridae) gill parasite of cultivated Piaractus mesopotamicus (Osteichthyes, Characidae) in Brazil. Efficacy and hematology

The present work evaluated the efficacy of mebendazole (MBZ) treatment against infections with the monogenean helminths Anacanthorus penilabiatus, gill parasites of young cultivated pacu, Piaractus mesopotamicus. A short-term bath treatment using 100, 200 and 500 mg MBZ/l of water for 10 and 30 min and a long-term-bath using 1, 10 and 100 mg MBZ/l of water for 24 h were utilized. Seven days after, fish were sacrificed and parasites counted. Concentrations of 500 and 200 mg MBZ/l for 10 and 30 min showed reduced efficacy (0.0 and 0.7%) and (14.2 and 11.0%), respectively. Nevertheless, 100 mg MBZ/l (10 min) and 10 nag MBZ/l (24 h) showed better efficacy (79.6 and 8 1.4%, respectively). Treatments for 24 h provoked increases in hematocrit, hemoglobin concentration and leukocyte number when compared to those untreated fish. Mebendazole treatments also provoked alterations in the defense blood cells especially in lymphocyte and thrombocyte numbers, when the fishes were submitted to 10 and 30 min baths.

Host-parasite relationships in paracoccidioidomycosis

A viewpoint of host-parasite relationships in paracoccidioidomycosis is presented. The characteristics of the fungus which are important to the host-parasite interaction are discussed. Aspects of inhibition of mycelium-to-yeast transformation by estrogens acting at receptors on the fungal wall and in the cytoplasm, and the role of polysaccharide components of the cell wall in virulence are reviewed. The natural mechanisms of host defense are also examined, including phagocytosis, complement system, natural-killer cells and genetic control of resistance and susceptibility. Finally, a discussion of granuloma morphogenesis and its relationship to the humoral and cellular anti-P. brasiliensis immune response is presented.

Theoretical Dynamics of Experimental Populations of Introduced and Native Blowflies (Diptera: Calliphoridae)

Equilibrium dynamics in experimental populations of Chrysomya megacephala (F.) and C. putoria (Wiedemann), which have recently invaded the Americas, and the native species Cochliomyia macellaria (F.), were investigated using nonlinear difference equations. A theoretical analysis of the mathematical model using bifurcation theory established the combination of demographic parameters responsible for producing shifts in blowfly population dynamics from stable equilibria to bounded cycles and aperiodic behavior. Mathematical modeling shows that the populations of the 2 introduced Chrysomya species will form stable oscillations with numbers fluctuating 3-4 times in successive generations. However, in the native species C. macellaria, the dynamics is characterized by damping oscillations in population size, leading to a stable population level.

The spatial dynamics of native and introduced blowflies (Dipt., Calliphoridae)

The spatial dynamics of three blowfly species was investigated using a spatially extended model of density-dependent population growth and the results indicate an overall stabilizing effect. Introduction of diffusive dispersal induced a quantitative effect of damping variation in population size on the route to a one-fixed point equilibrium in the native species, Cochliomyia macellaria. On the other hand, diffusive dispersal caused qualitative shifts in the dynamics of two invading species, Chrysomya megacephala and Chrysomya putoria. In both species diffusive dispersal can produce a qualitative shift from a two-point limit cycle to a one fixed-point dynamics. Quantitatively, dispersal also has the effect of damping oscillations in population size in the invading species.

Henneguya piaractus n.sp. (Myxozoa: Myxobolidae), a gill parasite of Piaractus mesopotamicus Holmberg, 1887 (Osteichthyes: characidae), in Brazil

Henneguya piaractus n.sp. was found from the gill filaments of Piaractus mesopotamicus (Characidae). Fishes were collected during a year from the reservoir at Centro de Aquicultura da UNESP, Jaboticabal, SP, Brazil. The prevalence of this parasite was 97.3% in the gills of farmed fish. Observations on spore development into the cysts were done. Studies with scanning electon microscopy were performed to observe the spore and cyst structure.

Mebendazole treatment against Anacanthorus penilabiatus (Monogenea, Dactylogyridae) gill parasite of cultivated Piaractus mesopotamicus (Osteichthyes, Characidae) in Brazil. Efficacy and hematology

The present work evaluated the efficacy of mebendazole (MBZ) treatment against infections with the monogenean helminths Anacanthorus penilabiatus, gill parasites of young cultivated pacu, Piaractus mesopotamicus. A short-term bath treatment using 100, 200 and 500 mg MBZ/1 of water for 10 and 30 min and a long-term bath using 1, 10 and 100 mg MBZ/1 of water for 24 h were utilized. Seven days after, fish were sacrificed and parasites counted. Concentrations of 500 and 200 mg MBZ/1 for 10 and 30 min showed reduced efficacy (0.0 and 0.7%) and (14.2 and 11.0%), respectively. Nevertheless, 100 mg MBZ/1 (10 min) and 10 mg MBZ/1 (24 h) showed better efficacy (79.6 and 81.4%, respectively). Treatments for 24 h provoked increases in hematocrit, hemoglobin concentration and leukocyte number when compared to those untreated fish. Mebendazole treatments also provoked alterations in the defense blood cells especially in lymphocyte and thrombocyte numbers, when the fishes were submitted to 10 and 30 min baths.

Antigens from Rhipicephalus sanguineus ticks elicit potent cell-mediated immune responses in resistant but not in susceptible animals

In the present study we compared the immunological reactions between Rhipicephalus sanguineus tick-infested susceptible (dogs and mice) and tick-resistant hosts (guinea pigs), elucidating some of the components of efficient protective responses against ticks. We found that T-cells from guinea pigs infested with adult ticks proliferate vigorously in the presence of concanavalin A (ConA), whereas ConA-induced cell proliferation of tick-infested mice and dogs was significantly decreased at 43.1 and 94.0%, respectively, compared to non-infested controls. Moreover, cells from mice and dogs submitted to one or three successive infestations did not exhibit a T-cell proliferative response to tick antigens, whilst cells from thrice tick-infested guinea pigs, when cultured with either a tick extract or tick saliva, displayed a significant increase in cell proliferation. Also, we evaluated the response of tick-infested mice to a cutaneous hypersensitivity test induced by a tick extract. Tick-infested mice developed a significant immediate reaction, whereby a 29.9% increase in the footpad thickness was observed. No delayed-type hypersensitivity (DTH) reaction was detected. Finally, the differential cell count at the tick attachment site in repeatedly infested mice exhibited a 6.6- and 4.1-fold increase in the percentage of eosinophils and neutrophils, respectively, compared to non-infested animals, while a decrease of 77.0-40.9 in the percentage of mononuclear cells was observed. The results of the cutaneous hypersensitivity test and the cellular counts at the tick feeding site for mice support the view that tick-infested mice develop an immune response to R. sanguineus ticks very similar to dogs, the natural host of this species of tick, but very different from guinea pigs (resistant host), which develop a DTH reaction in addition to a basophil and mononuclear cell infiltration at the tick-attachment site. In conclusion, saliva introduced during tick infestations reduces the ability of a susceptible animal host to respond to tick antigens that could stimulate a protective immune response. As a consequence, the animals present a lack of DTH response and disturbed cellular migration to tick feeding site, which can represent a deficient response against ticks. © 2003 Elsevier B.V. All rights reserved.